Hepatic macrophages express Chi3l1.

(A-D) Wildtype C57BL/6J mice were fed either a normal chow diet (NCD) or HFHC for 16 weeks. NPCs were isolated and subjected to BD Rhapsody scRNA sequencing. (A) Uniform manifold approximation and projection (UMAP) plots illustrate the clustering of NPCs in the livers of mice fed NCD and HFHC. Cell clusters are color-coded, with monocytes/macrophages clusters outlined. (B) UMAP plots depict the clustering of Monocytes/Macrophages in the livers of mice fed NCD and HFHC. Cell clusters are color-coded. (C) Dot plot displays the scaled gene expression levels of lineage-specific marker genes in different cell clusters. (D) Dot plot shows the scaled gene expression levels of Chil1 in the indicated cell clusters. (E) Immunofluorescent staining of TIM4 (white), F4/80 (red), Chi3l1 (green), and nuclear DAPI (blue) in liver sections of mice fed with either NCD or HFHC for 16 weeks, illustrating Chi3l1 expression in hepatic macrophages. Scale bar=20μm and 5μm (zoom). (F, G) Western blot analysis of Chi3l1 in either isolated Kupffer cells (KCs, F) or whole liver tissue (Liver, G) from mice fed either NCD or HFHC diet. n=2-3 mice/group. (H) mRNA expression levels of Chil1 in liver tissues of patients with metabolic dysfunction-associated fatty liver (MAFL) or with metabolic dysfunction-associated steatohepatitis (MASH) (GEO Datasets: GSE167523, GSE207310, GSE130970). No-MAFLD or Healthy individuals serve as controls. (I) The correlation between mRNA expression levels of Chil1 and MASLD activity score or fibrosis stage was analyzed (GEO Datasets: GSE130970). Representative images were shown in E. Mann-Whitney test was performed in F. Pearson’s correlation was performed in G. P value and r value are as indicated.

Deficiency of Chi3l1 in Kupffer cells promotes insulin resistance and hepatic lipid accumulation.

Chil1fl/fl and Clec4f△Chil1 mice were fed either a normal chow diet (NCD) or a high-fat, high-cholesterol (HFHC) diet for 16 weeks. (A, B) Body weight was recorded during HFHC diet feeding (A) and expressed as a percentage of initial body mass (B). (C) H&E (Upper panel) and oil red o staining (Lower panel) was performed to examine liver histology and hepatic lipid accumulation in in both genotypes after 16 weeks of NCD or HFHC diet. Scale bar = 20 µm. (D) Liver index (liver weight/body weight), ALT levels, and serum and liver Cholesterol or Triglyceride levels were measured in both genotypes after 16 weeks on NCD or HFHC diets. n=4-12 mice/group. (E, F) Intraperitoneal glucose tolerance test (IGTT) and insulin tolerance test (ITT) were performed after 16 weeks of NCD or HFHC feeding in both genotypes (n = 4–12 mice per group). Representative images were shown in (A, E). One-way ANOVA was performed in (A, B, D-F). P-value is as indicated.

ScRNA-seq reveals upregulated glucose metabolism-related transcripts in KCs, correlating with cell death signatures.

(A-D) Wildtype C57BL/6J mice were fed either a normal chow diet (NCD) or HFHC for 16 weeks. NPCs were isolated and subjected to BD Rhapsody scRNA sequencing. (A) Quantification of each cell cluster is presented. (B) KEGG analysis reveals the top 12 enriched pathways for up-regulated genes when comparing HFHC versus NCD in KCs, monocytes, and MoMFs, respectively. (C) Gene set variation analysis (GSVA) shows pathway activity for cell death, glucose metabolism, and cell proliferation in KCs, monocytes, and MoMFs of WT mice fed NCD or HFHC for 16 weeks, respectively. (D) The correlation between cell death and glucose metabolism pathways, based on GSVA score, is depicted.

Chi3l1 deficiency promote KCs death during MASLD.

(A) GSVA analysis showed the enrichment of cell death-related pathways in KCs from WT mice fed with either NCD or HFHC or Chil1-/- mice fed with HFHC. (B) Dot plot showing the scaled gene expression levels of Apoptosis-related genes and repressor genes in KCs from either WT or Chil1-/- fed with HFHC. (C) Strategy used to gate KCs (CD45+ F4/80hi CD11blow TIM4hi) and MoMFs (CD45+ F4/80low CD11bhi Ly6G− TIM4−) in the liver by flow cytometry. (D) Number of KCs and MoMFs /liver or gram(g) liver were statistically analyzed. n= 3-4 mice per group. (E) Immunofluorescent staining to detect TIM4(green), TUNEL (red), and nuclear DAPI (blue) in liver sections. Scale bar=20μm and 5μm(zoom). TUNEL+ TIM4+ cells/TIM4+ cells were statistically analyzed. n=4-6 mice/group. Representative images are shown in C, E. One-way ANOVA was performed in D. Two-tailed, unpaired student t-test was performed in E. P value is as indicated.

Molecular interaction between Chi3l1 and glucose.

(A) A comparison of chemical structures between glucose and chitin. (B) Prediction of Chi3l1-glucose interaction using STITCH database (http://stitch.embl.de). (C) Strategy for pulling down glucose-binding proteins in murine serum. (D) Biotin-conjugated glucose was incubated with murine serum from mice fed with HFHC for 16 weeks. Proteins bound to glucose were precipitated by streptavidin beads. Biotin or biotin-conjugated glucose plus glucose were used as negative controls. Western blot was performed to examine Chi3l1 in the precipitate. (E) Microscale thermophoresis assay to detect the interaction between recombinant mouse Chi3l1 (rChi3l1) and glucose. Kd=4.95±0.66mM. (F) Western blot to detect Chi3l1 expression in murine serum before and after HFHC feeding. n=3 mice/group.

Chi3l1 limits glucose uptake and protects hepatic macrophages from cell death.

(A) Following 12 h of glucose starvation, isolated KCs or BMDM were divided into two groups: one treated with no 2-NBDG and the other with 2-NBDG. Within each group, KCs or BMDM were further treated without or with recombinant murine Chi3l1 (rChi3l1) for 6 h. Glycogen aggregate formation labeled by 2-NBDG (Green) in KCs or BMDM was examined after counterstaining with nuclear DAPI (Blue). Scale bar=2μm. Area of 2-NBDG in KCs were quantified. (B) Following 12 h of glucose starvation, BMDM were treated with either no glucose or high glucose (25mM). Concurrently, BMDM were treated without or with rChi3l1 for 24 h under each condition. glycogen aggregate formation in BMDM was detected using immunofluorescence staining for Stbd1 (red) and nuclear DAPI (blue). Scale bar = 10 μm. (C and D) BMDM cells were treated without or with rChi3l1 for 24 h and subjected to Seahorse metabolic analysis to measure the extracellular acidification rate (ECAR). (E and F) KCs were treated without (blank) or with either Isopropyl alcohol(Iso) or 800uM palmitic acid (PA) or 100ng rChi3l1 with 800 uM PA for 24 h. Western blot was performed to detect cleaved caspase 3 (Cl-Casp3) in E. Calcein/PI staining was quantified to detect cell viability in F. Scale bar=50μm. (G) Measurement of 2-NBDG (a fluorescent glucose analog) uptake by KCs in vivo. WT and Chil1-/- mice, either untreated or supplemented with rChi3l1, were injected intraperitoneally with 12 mg/kg 2-NBDG. After 45mins, KCs were isolated and glucose uptake assessed by spectrophotometry. (H) Representative immunofluorescence images of liver sections stained for TIM4 (red) and 2-NBDG uptake (green) to visualize glucose uptake by KCs in situ. Scale bar = 10 µm (zoom). Quantification is shown as the percentage of TIM4+ cells that are also 2-NBDG+. Representative images were shown in A, B, H. One-way ANOVA was performed in A, F, G, H. Two-tailed, unpaired student t-test was performed in D. P value is as indicated.

Differential regulation of KCs and MoMFs fate by Chi3l1-glucose interaction.

KCs maintain a high-glucose activation state, while MoMFs exhibit a relatively low-glucose metabolic program. Chi3l1-glucose binding inhibits glucose uptake in KCs, thereby delaying KCs death and alleviating MASLD progression and metabolic dysfunction. In contrast, although Chi3l1-glucose binding similarly inhibits glucose uptake in MoMFs, their low basal glucose metabolism renders them resistant to this metabolic perturbation, resulting in minimal impact on MASLD pathogenesis.